The advantages provided by the zebrafish neuromuscular synapse have led to discoveries that have greatly advanced our basic understanding of neurotransmission and neuromuscular diseases. Driving all our studies has been our development of paired motoneuron muscle patch clamp recordings, which has only been possible in zebrafish. We recently used this methodology to explore frequency-dependent depression, one of the most prevalent forms of synaptic plasticity, and obtained evidence for heterogeneity among release sites. The existence of functionally distinct classes of release sites remains an unexplored territory among vertebrates, calling for development of probes adequate to detect their function. Also unique to zebrafish neuromuscular junction is a small number of release sites that are well separated from one another. To probe each of these release sites independently we created a transgenic line wherein the calcium indicator GCaMP6f is fused to postsynaptic rapsyn. Due to the high calcium permeability of the nicotinic receptor, we have been able to optically track release of single quanta from individual motoneuron release sites. With this powerful detector, we now outline a series of experiments that will test for release site heterogeneity and its role in synaptic depression. We also utilize optical quantal analysis to further address an outstanding question asking whether the synchronous, asynchronous and spontaneous modes of synaptic transmission work through the same or different release sites. Finally, to identify the structural counterparts of functional heterogeneity, we are collaborating with Christian Stigloher, an expert in high-resolution tomographic analysis of zebrafish neuromuscular synapses. Individual fast and slow synapses will be identified on the basis of optical quantal analysis and subjected to tomographic reconstruction to test for differences in cytomatrix components of the active release site. Only through the many unique features offered by this vertebrate cholinergic synapse is it possible to perform the proposed experiments to decipher synaptic depression and associated use dependent fatigue, common to almost all myasthenias.